Peptide linkers for polypeptide compositions and methods for using same

ABSTRACT

Disclosed herein are novel peptide linkers and polypeptide compositions comprising the linkers (e.g., chimeric polypeptides) and methods of using the polypeptide compositions. The compositions and methods are particularly useful for targeting/delivering a polypeptide or protein of interest (e.g., a therapeutic polypeptide) to a cell, tissue or organ of interest in order to treat various diseases or disorders (e.g., lysosomal storage disorders).

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/449,225 filed on Mar. 4, 2011, and is a continuation-in-part of U.S.application Ser. No. 13/168,969 filed on Jun. 25, 2011 now abandoned andInternational Application No. PCT/US2011/041928 filed on Jun. 25, 2011,the entire teachings of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present inventions are directed to novel peptide linkers andpolypeptide compositions comprising such linkers (e.g., chimericpolypeptides) and methods of using and preparing the same.

BACKGROUND OF THE INVENTION

Lysosomal storage disorders represent a group of more than forty rareand inherited metabolic disorders caused by the deficiency or inactivityof specific lysosomal enzymes. In particular, lysosomal storagedisorders are caused by the deficiency or inactivity of the lysosomalenzymes which catalyze the stepwise metabolism of lipids or complexglycoproteins known as glycosaminoglycans. As a result of this metabolicdeficiency, metabolic precursors progressively accumulate in the cells,tissues and, in particular, the cellular lysosomes of affected subjects.This protein accumulation causes a permanent, progressive cellulardamage which affects the appearance, physical abilities, organ andsystem functioning and, in most cases, the mental development ofaffected subjects. Although the enzyme deficiencies affect every tissue,a patient's clinical expression may frequently vary depending, forexample, on the degree of enzyme deficiency or impairment. The lysosomalstorage disorder may also be associated with some degree of neuronalcell loss, predominantly resulting in neurological symptoms, including,mental retardation, severe motor impairments, physical disability, adecreased lifespan and/or combinations of the foregoing.

There are no cures for the lysosomal storage disorders, and treatment isoften palliative, offered to subjects primarily to improve their qualityof life. Enzyme replacement therapy (ERT) has been a useful therapeuticoption for subjects with lysosome storage disorders. ERT generallyinvolves the parenteral administration of natural orrecombinantly-derived proteins and/or enzymes to a patient. Approvedtherapies are administered to patients intravenously and are generallyeffective in treating the somatic or peripheral symptoms of theunderlying enzyme deficiency. To effectively treat lysosomal storagedisorders, the administered therapeutic agent (e.g., the deficientlysosomal enzyme) must distribute into the affected cells and tissuesafter being infused into a patient's bloodstream.

To achieve distribution of the requisite enzymes into affected cells andtissues, the enzymes are generally targeted to specific cell-surfacereceptors that transport the enzymes into the cells throughreceptor-mediated endocytosis. For example, in Gaucher's disease, thedeficient enzyme, glucocerebrosidase, is targeted to the appropriatecells through the binding of exposed mannose residues on the enzyme tothe mannose receptor, which is abundantly expressed on target cells(reticuloendotheilial cells). In cells that lack the mannose receptor,use of the insulin-like growth factor/cation-independentmannose-6-phosphate receptor (IGF-II/CI-MPR) has been proposed fordelivery of deficient lysozymes to cells (Kornfeld, S., 1987 Biochem SocTrans 18:367-374). The IGF-II/CI-MPR receptor is present on the surfaceof many mammalian cell types and thus provides a means by which totarget proteins containing the receptor ligand (e.g., IGFII or mannose-6phosphate) to a wide variety of cells and tissues, including the centralnervous system. However, despite some knowledge of how to target missinglysosomal enzymes to appropriate tissues, there are still no effectivetherapies for many lysosomal storage disorders (e.g., Sanfilipposyndrome, Farber's disease, and the like). Thus, there remains a need inthe art for compositions, particularly compositions that can beadministered parenterally, and methods useful for directing agents tothe necessary tissues to treat diseases (e.g., lysosomal storagediseases).

SUMMARY OF THE INVENTION

There is a need for compositions and methods that facilitate thetransport and delivery of functional therapeutic agents (e.g., proteins,polypeptides) to the desired tissues. Such compositions and methods maybe useful in the treatment of a number of diseases or disorders and, inparticular, in the treatment of lysosomal storage disorders, likeSanfilippo disease.

Described herein are novel compositions comprising peptide linkers,polypeptide compositions comprising polypeptides joined by the peptidelinkers and related polynucleotides, vectors, cells and pharmaceuticalcompositions. Described linker sequences operably join twopeptides/polypeptides of interest such that the expression and activity(e.g., receptor binding and/or enzyme activity) of the polypeptidesconnected by the linkers are durable and optimal. The polypeptidecompositions comprising the peptide linkers facilitate the targeteddelivery of polypeptides/proteins of interest to particular cells and/ortissues.

Accordingly, an embodiment of the invention provides for a polypeptidecomposition comprising a first peptide/polypeptide, a secondpeptide/polypeptide and a linker comprising one or more sequential ortandem repeats of the amino acid sequence of SEQ ID NO. 1(GAPGGGGGAAAAAGGGGG) disposed between the first peptide and the secondpeptide. In some embodiments, the linker of the polypeptide compositioncomprises three sequential or tandem repeats of SEQ ID NO. 1 and, insome embodiments, the linkers further comprise the amino acid sequenceglycine alanine proline (GAP) at the 3′ end of SEQ ID NO. 1. In otherembodiments, one or more alanine residues of the linkers can besubstituted with one or more serine residues. In certain embodiments,the first peptide of the polypeptide composition comprises the aminoacid sequence of SEQ ID NO. 4. In still other embodiments the secondpeptide comprises a receptor binding domain and, in further embodiments,the second peptide comprises the amino acid sequence of SEQ ID NO. 6.

In certain embodiments, a polypeptide composition comprises a firstpeptide comprising the amino acid sequence of SEQ ID NO. 4; a secondpeptide comprising the amino acid sequence of SEQ ID NO. 6; and a linkercomprising one or more sequential or tandem repeats of the amino acidsequence of SEQ ID NO. 1 disposed between the first peptide and thesecond peptide. In some embodiments, the linker comprises threesequential repeats of SEQ ID NO. 1. In other embodiments, the linkerscan further comprise the amino acid sequence glycine alanine proline(GAP) at the 3′ end of the SEQ ID NO. 1. In still other embodiments, oneor more alanine residues of the linkers are substituted with one or moreserine residues.

In some embodiments, a polypeptide composition comprises a first peptidecomprising the amino acid sequence of SEQ ID NO. 4; a second peptidecomprising the amino acid sequence of SEQ ID NO. 6; and a linkercomprising the amino acid sequence of SEQ ID NO. 2 disposed between thefirst peptide and second peptide. In certain embodiments, one or morealanine residues of the linker are substituted with one or more serineresidues.

The invention also provides for peptide/polypeptide linkers which aredescribed herein. For example, in some embodiments, a polypeptide linkercomprises 18 contiguous amino acid residues, wherein the linkercomprises 1 proline residue within the first five amino acid residues ofthe linker and 17 amino acid residues selected from the group consistingof one or more glycine residues and one or more alanine residues. Inother embodiments, the 3′ end of the polypeptide linker furthercomprises three contiguous amino acids comprising 1 proline residue andtwo amino acid residues selected from the group consisting of glycineand alanine. In some embodiments, one or more alanine residues of thelinker are substituted with one or more serine residues.

In some embodiments, a polypeptide linker comprises 21 contiguous aminoacid residues wherein the linker comprises a first proline residue thatis within the first five amino acid residues of the linker; 19 aminoacid residues selected from the group consisting of one or more glycineresidues and one or more alanine residues; and a second proline residuethat is within the last five amino acids of the linker.

In certain embodiments, one or more alanine residues of the linkers aresubstituted with one or more serine residues. In still otherembodiments, the polypeptide linkers comprise two times as many glycineand serine residues as alanine residues.

In some embodiments, the polypeptide linker consists of the amino acidsequence of SEQ ID NO. 7 (GGGGGAAAAAGGGGG).

In other embodiments, the invention provides for polypeptide linkercompositions that comprise one or more sequential repeats of apolypeptide linker consisting of the amino acid sequence of SEQ ID NO.7. In other embodiments, the 5′ end of the polypeptide linkercomposition further comprises three contiguous amino acids comprisingone proline residue and two amino acid residues selected from the groupconsisting of glycine and alanine.

In still other embodiments, a polypeptide linker consists of the aminoacid sequence of SEQ ID NO. 1. In certain embodiments, the inventionprovides for a polypeptide linker composition comprising one or moresequential repeats of a polypeptide linker consisting of SEQ ID NO. 1.

In further embodiments, the 3′ end of the above polypeptide linkercompositions further comprises three contiguous amino acids comprisingone proline residue and two amino acid residues selected from the groupconsisting of glycine and alanine. In certain embodiments, the threecontiguous amino acid residues comprise the amino acid sequence glycinealanine proline (GAP).

In some embodiments, a polypeptide linker consists of the amino acidsequence of SEQ ID NO. 2(GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP).

In certain embodiments, the invention provides for a polypeptidecomposition comprising a first peptide; a second peptide; and apolypeptide linker disposed between the first peptide and the secondpeptide, wherein the linker comprises any of the aforementionedpolypeptide linkers or polypeptide linker compositions.

In other embodiments, the invention also provides for polynucleotidesthat encode the polypeptide linkers and/or polypeptide compositionsdescribed herein. Thus, some embodiments provide a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO.1; SEQ ID NO. 2; or one or more sequential repeats of SEQ ID NO. 1. Insome embodiments, one or more alanine residues of the polypeptide aresubstituted with one or more serine residues and the polynucleotideencodes for the one or more substituted serine residues.

In other embodiments, a polynucleotide encodes a polypeptide compositioncomprising the amino acid sequence of a first peptide; a second peptide;and a linker comprising one or more sequential repeats of the amino acidsequence of SEQ ID NO. 1 disposed between the first peptide and thesecond peptide. In certain embodiments, the linker of the polypeptidecomposition comprises three sequential or tandem repeats of SEQ ID NO.1.In still other embodiments, one or more alanine residues of the linkerof the polypeptide compositions are substituted with one or more serineresidues, and the polynucleotide encodes the one or more substitutedserine residues. In further embodiments of the foregoingpolynucleotides, the 3′ end of the linker of the polypeptidecompositions further comprises the amino acid sequence glycine alanineproline (GAP), and the polynucleotide encodes the GAP amino acidsequence.

In certain embodiments, a polynucleotide encodes first peptide of thepolypeptide composition that comprises the amino acid sequence of SEQ IDNO. 4. In other embodiments, the polynucleotide encodes a second peptideof the polypeptide composition that comprises a receptor binding domainand, in some embodiments, the second peptide comprises the amino acidsequence of SEQ ID NO. 6.

Also described herein are expression vectors comprising theaforementioned polynucleotides which encode the polypeptide compositionsdescribed herein. Other embodiments described herein relate torecombinant cells comprising the foregoing polynucleotides andexpression vectors.

In addition, the invention provides for pharmaceutical compositionscomprising the polypeptide compositions described herein and apharmaceutically acceptable carrier. For example, in some embodiments,the pharmaceutical composition comprises polypeptide compositionscomprising a first peptide comprising the amino acid sequence of SEQ IDNO. 4 and a second peptide comprising the amino acid sequence of SEQ IDNO. 6. In some of these embodiments, the linker disposed between thefirst peptide and second peptide comprises one or more sequential ortandem repeats of SEQ ID NO. 1 (e.g., one, two, three, four, five, six,seven, eight, nine, ten or more sequential or tandem repeats of SEQ IDNO. 1), and in other of these embodiments, the linker comprises theamino acid sequence of SEQ ID NO. 2.

In certain embodiments, the invention also provides for pharmaceuticalcompositions comprising the polynucleotides described herein and apharmaceutically acceptable carrier. The invention further provides forpharmaceutical compositions comprising the recombinant cells describedherein and a pharmaceutically acceptable carrier.

Also described herein are methods of producing a polypeptidecomposition, the method comprising culturing the recombinant cellsdescribed herein under conditions suitable for the expression of thepolypeptide.

Methods of delivering a therapeutic polypeptide to a subject in needthereof are also described herein. Thus, some embodiments disclosedherein are methods of delivering a therapeutic polypeptide to a subjectin need thereof comprising administering to the subject any of theaforementioned polypeptide compositions described herein.

In other embodiments, a method of delivering a therapeutic polypeptideto a subject in need thereof comprises administering to the subject apolypeptide composition comprising a first peptide comprising the aminoacid sequence of SEQ ID NO. 4; a second peptide comprising the aminoacid sequence of SEQ ID NO. 6; and a linker comprising one or moresequential repeats of the amino acid sequence of SEQ ID NO. 1 disposedbetween the first peptide and the second peptide. In other embodimentsof the method, the linker further comprises GAP at the 3′ end of SEQ IDNO. 1. In certain embodiments of the method, the linker comprises theamino acid sequence of SEQ ID NO. 2. In still other embodiments of themethods, one or more alanine residues of the linkers are substitutedwith one or more serine residues.

In some embodiments, a method of delivering a therapeutic polypeptide toa subject in need thereof comprises administering to the subject one ormore of the above-mentioned expression vectors described herein. Certainother embodiments relate to methods of delivering a therapeuticpolypeptide to a subject in need thereof comprising administering to thesubject one or more of the aforementioned recombinant cells describedherein.

Also described herein are methods of treating a lysosomal storagedisease, the method comprising, in some embodiments, administering to asubject in need thereof an effective amount of any one or theaforementioned pharmaceutical compositions described herein (comprisinge.g., polypeptide compositions, polynucleotides and/or host cellsdescribed herein). In certain embodiments, the lysosomal storage diseaseis Sanfilippo syndrome.

Still other embodiments described herein relate to methods of treatingSanfilippo syndrome. In some embodiments, the method comprisesadministering to a patient in need thereof an effective amount of theaforementioned pharmaceutical compositions described herein.

In other embodiments, a method of treating Sanfilippo syndrome comprisesadministering to a patient in need thereof an effective amount of apharmaceutical composition comprising a first peptide comprising theamino acid sequence of SEQ ID NO. 4; a second peptide comprising theamino acid sequence of SEQ ID NO. 6; and a linker comprising one or moresequential repeats of SEQ ID NO. 1 disposed between the first peptideand the second peptide. In certain embodiments of the method, the linkerfurther comprises the amino acid sequence gap at the 3′ end of SEQ IDNO. 1. In other embodiments of the method, the linker comprises theamino acid sequence of SEQ ID NO. 2.

In certain embodiments of the methods, the pharmaceutical compositionsare administered parenterally.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of a peptide linkercomprising a glycine alanine praline (GAP) sequence joined to a glycinealanine glycine (GAG) repeat sequence (SEQ ID NO. 1).

FIG. 2 illustrates the amino acid sequence of a peptide linkercomprising three sequential or tandem repeats of SEQ ID NO. 1 joined atthe 3′ end to a GAP sequence (SEQ ID NO. 2).

FIG. 3A illustrates the α-N-acetylglucosaminidase-insulin-like growthfactor (NaGlu-IGFII) construct joined by a GAP linker. FIG. 3Billustrates a NaGlu-IGFII construct joined by the linker of SEQ ID NO. 2(NaGlu-GAG₃-IGFII).

FIG. 4A illustrates the nucleotide sequence of human NaGlu (SEQ ID NO.3). FIG. 4B illustrates the amino acid sequence of human NaGlu (SEQ IDNO. 4).

FIG. 5A illustrates the nucleotide sequence of human IGFII (SEQ ID NO.5) FIG. 5B illustrates the amino acid sequence of human IGFII (SEQ IDNO. 6).

FIG. 6 illustrates the amino acid sequence of a peptide linkercomprising a GAG repeat sequence (SEQ ID NO. 7).

FIG. 7 illustrates the amino acid sequence of the NaGlu-IGFII constructillustrated in FIG. 3A (SEQ ID NO. 8).

FIG. 8 illustrates the amino acid sequence of the NaGlu-IGFII constructillustrated in FIG. 3B (SEQ ID NO. 9).

FIG. 9 illustrates a comparison of the activity levels of two differentNaGlu-IGFII protein constructs (NaGlu-GAG₃-IGFII and NaGlu-IGFII) inHT1080 cells and demonstrates that, compared to wild-type NaGlu,NaGlu-GAG₃-IGFII has very high levels of activity, while NaGlu-IGFII hasvery little.

FIG. 10 illustrates the expression of NaGlu-GAG₃-IGFII and NaGlu-IGFIIpolypeptides by western blot and shows that the NaGlu-IGFII proteinunderwent degradation while wild-type NaGlu and NaGlu-GAG₃-IGFII didnot.

FIG. 11 illustrates the uptake of the NaGlu-GAG₃-IGFII polypeptide byhuman fibroblast cells (HF1156) and demonstrates that NaGlu-GAG₃-IGFIIwas readily taken-up by human cells.

FIG. 12 illustrates the amino acid sequence of a peptide linker (SEQ IDNO. 10).

FIG. 13 illustrates the amino acid sequence of a peptide linker (SEQ IDNO. 11).

FIG. 14 illustrates the amino acid sequence of a peptide linker (SEQ IDNO. 12).

DETAILED DESCRIPTION OF THE INVENTION

Compositions are described herein that provide a means to make (e.g.,design, engineer) chimeric or fusion polypeptides. The polypeptidecompositions can also provide means of facilitating the delivery ofagents (e.g., polypeptides/peptides, proteins and/or enzymes) to cells,tissues or organs of interest. In particular, the compositions andmethods can be used to selectively deliver agents to an appropriatetissue of a subject in need thereof, thereby treating a disease ordisorder. These therapeutic compositions can be polynucleotides orpolypeptides that comprise a therapeutic agent (e.g., protein/enzyme)joined to a targeting agent (e.g., cell receptor ligand protein) by alinker sequence that allows for the proper expression, folding andactivity of the therapeutic and/or targeting agent. In some aspects, thetherapeutic composition comprises a lysosomal protein or enzymeconnected by the linker sequence to a cell-surface receptor ligandprotein. The therapeutic polypeptide composition can be used to treatdisorders such as lysosomal storage diseases.

As used herein, the phrase “lysosomal storage disorder” or lysosomalstorage disease” refers to a class of inherited diseases related to theaberrant expression of or deficiency of one or more lysosomal enzymes.These enzyme deficiencies result in detrimental accumulation ofmetabolic products in the lysosomes of affected subjects. Representativelysosomal storage disorders include aspartylglucosaminuria, cholesterylester storage disease, cystinosis, Danon disease, Fabry disease,Farber's disease, fucosidosis, falactosialidosis types I/II, Gaucherdisease types 1, 2, 3, globoid cell leucodystrophy/Krabbe disease,glycogen storage disease II/Pompe disease, GM1-gangliosidosis typesI/III, GM2-gangliosidosis type I/Tay-Sachs disease, GM2-gangliosidosistype II/Sandhoff disease, α-mannosidosis types I/II, β-mannosidosis,metachromatic leukodystrophy (MLD), mucolipidosis type I/sialidosistypes I/II, mucolipidosis types II/III, mucolipidosis type IIIpseudo-Hurler polydystrophy, mucopolysaccharidosis (e.g. types I, II,IIIA, IIIB, IIIC, IIID, IVA, IVB, VI, VII and IX), multiple sulphatasedeficiency, neuronal ceroid lipofuscinosis (e.g. Batten, infantile, lateinfantile and adult), Niemann-Pick disease (e.g. types A, B, C1, C2),Schindler disease types I/II, sialic acid storage disease, Sanfilippodisease, and Wolman's disease (acid lipase deficiency). In one aspect ofthe invention, the compositions comprise a therapeutic agent whosedeficiency is linked to a lysosomal storage disorder.

Polypeptide composition and polynucleotides encoding the polypeptidecompositions are described herein, in which the polypeptide compositionscomprise a first and second peptide/polypeptide, connected by a linkersequence disclosed herein. The inventors have surprisingly found that alinker comprising one or more sequential or tandem repeats of SEQ ID NO.1 (GAPGGGGGAAAAAGGGGG), connecting two protein sequences (e.g., a firstpolypeptide and a second polypeptide) results in the production of apolypeptide that is well-expressed and highly active (e.g., biologicallyactive). As used herein, the term “polypeptide” or “peptide” refers apolymer of amino acid residues typically joined exclusively by peptidebonds, that can be produced naturally (e.g., isolated, essentiallypurified or purified) or synthetically (e.g., by chemical synthesis). Apolypeptide produced by expression of a non-host DNA molecule is a“heterologous” peptide or polypeptide. An “amino acid residue”comprising the polypeptide can be a natural or non-natural amino acidresidue linked by peptide bonds and/or bonds different from peptidebonds. The amino acid residues can be in D-configuration orL-configuration. In some aspects, the polypeptides referred to hereinare proteins, peptides or fragments thereof produced by the expressionof recombinant nucleic acid. In some embodiments, the polypeptidecompositions described herein comprise two polypeptides connected by alinker sequence (e.g., SEQ ID NO. 2), in which one of the twopolypeptides is a peptide that can be administered to treat a disease ora disorder (e.g., a therapeutic peptide) and the other polypeptide ispeptide that can be used to deliver a therapeutic peptide to a targetcell, tissue or organ (e.g., a targeting peptide).

The linker or polypeptide linker described herein refers to a peptidesequence designed to connect (e.g., join, link) two protein sequences,wherein the linker peptide sequence is typically not disposed betweenthe two protein sequences in nature. In the context of the presentinvention, the phrase “linked” or “joined” or “connected” generallyrefers to a functional linkage between two contiguous or adjacent aminoacid sequences to produce a polypeptide that generally does not exist innature. In certain embodiments, linkage may be used to refer to acovalent linkage of, for example, the amino acid sequences of the one ormore therapeutic peptide agents and the one or more targeting agents(e.g., binding or receptor ligand peptides). Generally, linked proteinsare contiguous or adjacent to one another and retain their respectiveoperability and function when joined. Peptides comprising the chimericpolypeptides disclosed herein are linked by means of an interposedpeptide linker comprising one or more amino acids. Such linkers mayprovide desirable flexibility to permit the desired expression, activityand/or conformational positioning of the chimeric polypeptide. A typicalamino acid linker is generally designed to be flexible or to interpose astructure, such as an alpha-helix, between the two protein moieties. Alinker can be fused to the N-terminus or C-terminus of a polypeptideencoding a lysosomal enzyme, or inserted internally. A linker is alsoreferred to as a spacer.

The linker peptide sequence can be of any appropriate length to connectone or more proteins of interest and is preferably designed to besufficiently flexible so as to allow the proper folding and/or functionand/or activity of one or both of the peptides it connects. Thus, thelinker peptide can have a length of no more than 3, no more than 5, nomore than 10, no more than 15, no more than 20, no more than 25, no morethan 30, no more than 35, no more than 40, no more than 45, no more than50, no more than 55, no more than 60, no more than 65, no more than 70,no more than 75, no more than 80, no more than 85, no more than 90, nomore than 95 or no more than 100 amino acids. In some embodiments, thelinker peptide can have a length of at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least12, at least 15, at least 18, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50 amino acids. In someembodiments, the linker comprises at least 10 and no more than 60 aminoacids, at least 10 and no more than 55 amino acids, at least 10 and nomore than 50 amino acids, at least 10 and no more than 45 amino acids,at least 10 and no more than 40 amino acids, at least 10 and no more 35amino acids, at least 10 and no more than 30 amino acids, at least 10and no more than 25 amino acids, at least 10 and no more than 20 aminoacids or at least 10 and no more than 15 amino acids. In certainembodiments, the linker comprises 12 to 57 amino acids, and inparticular embodiments, comprises 57 amino acids. In a polypeptidecomposition comprising a linker, the 5′ end (e.g., terminus) of thelinker peptide sequence (e.g., amino acid sequence) is adjacent to andcovalently linked to the 3′ end of one protein sequence (e.g.,full-length protein or protein domain, fragment or variant) and,further, the 3′ end of the linker amino acid sequence is adjacent to andcovalently linked to the 5′ end of another protein sequence. Polypeptidecompositions produced in this manner are commonly referred to a fusionor chimeric protein/polypeptides and typically are made by theexpression (e.g., transcription, translation) of nucleic acid sequencesencoding the polypeptide compositions, in the appropriate system. Meansby which to make fusion and/or chimeric polypeptides are well-known inthe art (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Harbor Laboratory, 1992) New York whichis incorporated by reference herein in its entirety).

In certain embodiments, the linker amino acid sequence is comprised ofglycine, alanine and/or serine amino acid residues. The inventors havediscovered that simple amino acids (e.g., amino acids with simple sidechains (e.g., H, CH₃ or CH₂OH) and/or unbranched) are advantageous foruse in a peptide linker as the lack of branched side chains on theseamino acids provides greater flexibility (e.g., two-dimensional orthree-dimensional flexibility) within the linker and, accordingly,within a polypeptide composition. Further, the inventors have found thatalternating the glycine, alanine and/or serine residues provides evenmore order and greater flexibility with in the linker. The amino acidscan alternate/repeat in any manner consistent with the linker remainingfunctional (e.g., resulting in expressed and/or active polypeptide(s)).In any of the linkers, the alanine amino acid residues can besubstituted with serines. Thus, the amino acids in the liker can repeatevery one (e.g., GAGA, GSGS), every two (e.g., GGAAGGAA, GGSSGGSS),every three, every four, every five, every 6, every 7, every 8, every 9or every 10 or more amino acids, or the amino acids can repeat in anycombination of the foregoing. In certain embodiments, the amino acidsrepeat every five amino acids and the linker consists of one or moreglycine alanine glycine repeats. For example, the peptide linker canconsist of a GGGGGAAAAAGGGGG (SEQ ID NO. 7) or GGGGGSSSSSGGGGG (SEQ IDNO: 10) repeat.

In addition, the inventors have discovered that placing a prolineresidue within the first (e.g., 5′ end) and/or last (e.g., 3′ end) fiveamino acids of the linker provides for additional benefit within thelinker. For example, a linker or spacer can be GAP (SEQ ID NO. 11) orGGGGGP (SEQ ID NO. 12). Not to be bound by theory, it is believed that,unlike glycine, alanine and serine which are flexible amino acids,proline, whose cyclic side chain results in inflexibility, may result ina kink near the end(s) of the otherwise flexible linker, and therebykeep the polypeptides connected by the linker appropriately separated.Thus, the linker amino acid sequence can have a proline in the first,second, third, fourth or fifth amino acid residue and/or a prolinewithin the last, second to last, third to last, fourth to last or fifthto last amino acid residue within the linker. In certain embodiments,the peptide linker can comprise 18 contiguous amino acid residues inwhich one of the amino acid residues is a proline that is located at anyone of the first five amino residues of the linker, and the remaining 17amino acid residues are comprised of glycine and alanine residues (e.g.,one or more glycine and one or more alanine residues). The glycine andalanine amino acid residues of the linker can comprise any combinationof glycine and alanine residues, including the aforementioned glycinealanine glycine repeats. The linker can further comprise threecontiguous amino acids comprising a second proline, a glycine residueand/or an alanine residue, to produce a peptide linker comprising 21amino acids. The second proline may also be any one of the last fiveamino acids in the linker amino acid sequence.

The foregoing peptide linkers can be flanked by one or more amino acidsequences that are encoded by a desired restriction endonuclease site orsites. Numerous endonuclease cleavage sites (e.g., EcoRI, BamHI,HindIII, AscI sites and the like) are well-known in the art, and theselection of which cleavage sites to include in the linker (and/orpolypeptide(s)) nucleic acid sequence is best determined by the skilledartisan, the site generally being chosen with regard to the respectivenucleic acid sequences being linked. The endonuclease restriction sitescan be the same site on each end of the linker sequence or differentrestriction sites as needed and/or desired. In some embodiments, theglycine alanine glycine amino acid repeats of the linker are flanked byglycine alanine proline (GAP) amino acid sequences at the 5′ and/or 3′end of the linker amino acid sequence (e.g., SEQ ID NO. 2). The GAPsequence in this instance is encoded for by nucleic acid sequence thatrepresents an AscI restriction endonuclease site. In some embodiments,the linker amino acid sequence comprises SEQ ID NO. 1(GAPGGGGGAAAAAGGGGG). In other embodiments, the linker amino acidsequence comprises one or more (e.g., 1, 2, 3, 4 or more) sequentialrepeats of SEQ ID NO. 1. The inventors have discovered that even onerepeat of SEQ ID NO. 1 improves the linker functionality with three andfour repeats of SEQ ID NO. 1 being most effective in allowing expressionand/or activity of the linked polypeptides. In certain embodiments, theone or more sequential repeats of SEQ ID NO. 1 are further comprised ofa gap sequence at the 3′ end/terminus of the linker's amino acidsequence. In some embodiments, the linker amino acid sequence comprisesSEQ ID NO. 2(GAPGGGGGAAAAAGGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP).

In some embodiments, a suitable linker or spacer may contain a sequenceat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to the sequence of SEQ ID NO. 2.

In the polypeptide compositions described herein, the two polypeptides(e.g., a first polypeptide and a second polypeptide) can berecombinantly joined by any of the linker polypeptides described above,with the linker disposed between the two polypeptides. For example, incertain embodiments, the polypeptides or compositions comprise a firstand a second polypeptide recombinantly joined by a linker comprising SEQID NO. 1 or SEQ ID NO. 2. The two polypeptides can be any amino acidsequences including full-length proteins, protein fragments or portions,functional protein fragments or portions, functional protein domains andthe like, of either two different proteins or the same protein. As usedherein, “functional fragment” or “portion” is intended to refer to lessthan the entire mature or native protein which is sufficient to retainone or more of the desired biological activities of the mature or nativeprotein (e.g., sufficient to retain a therapeutic or ameliorativebiological activity with respect to a disorder to be treated). Thus,amino acid sequences or polypeptides can be modified, for example,polypeptide sequences into which amino acids have been inserted, deletedand/or substituted in such a manner that the modifications do notsubstantially interfere with the polypeptide's ability to encode afunctional agent.

In some embodiments, one protein of the polypeptide composition is apeptide having a desired activity, while the other polypeptide deliversor targets the polypeptide having a desired activity to a specific cellor tissue. As used herein, the phrase targeting ligand or bindingpeptide refers to an amino acid sequence which serves to direct anddeliver an agent (e.g., protein, polypeptide) to a specific site for thedesired activity. In particular embodiments, the desired activity of oneof the polypeptides is a therapeutic or prophylactic activity (e.g.,treatment, replacement, inhibition, prevention, enhancement, reductionor amelioration). For example, in some embodiments, the polypeptidecompositions described herein comprise one or more enzymes and/orproteins that are deficient in a lysosomal storage disease/disorder. Forinstance, the disclosed compositions may comprise one or moretherapeutic agents comprising or consisting of an amino acid sequencederived from one or more of aspartylglucosaminidase, acid lipase,cysteine transporter, Lamp-2, α-galactosidase A, lipoprotein lipase(LPL), ceramidase, α-L-fucosidase, β-hexosaminidase A, β-glucoronidase,GM2 ganglioside activator protein, α-D-mannosidase, β-D-mannosidase,arylsulphatase A, saposin B, neuraminidase, α-N-acetylglucosaminidase,phosphotransferase, phosphotransferase, L-iduronidase,iduronate-2-sulphatase, idursulfase, heparan-N-sulphatase, heparinsulfamidase, α-N-acetylglucosaminidase, N-acetyltransferase,N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,β-galactosidase, N-acetylgalactosamine 4-sulphatase, N-acetylglucosamine6-sulfatase, hyalurono-glucosaminidase, multiple sulphatases, palmitoylprotein thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,α-galactosidase B, sialic acid, and functional fragments, subunits andcombinations of the above. In certain embodiments, one of the proteins(e.g., the therapeutic protein) of a polypeptide composition comprisesN-acetyl-alpha-glucosaminidase (NaGlu), particularly human NaGlu or afunctional portion, fragment, variant, mutant or derivative of NaGlu.Loss of the lysosomal enzyme NaGlu is believed to be responsible for thelysosomal storage disorder, Sanfilippo syndrome.

In some embodiments, one of the polypeptides of the polypeptidecomposition comprises a cell-surface receptor ligand and, in particularembodiments, the polypeptide is IGFII, one of the ligands of theIGFII/cation-independent mannose 6-phosphate receptor (IGFII/CI-MPR).The IGFII/CI-MPR recognizes mannose 6-phosphate (Man6-P) moieties addedto oligosaccharides on newly synthesized lysosomal enzymes in mammaliancells. As the Man6-P interaction with the IGFII/CI-MPR regulates normalintracellular trafficking that brings newly synthesized enzymes to thelysosome, IGFII/CI-MPR is thought to be a receptor mechanism that couldbe used to deliver the lysosomal enzymes to cells. The above-describedcompositions would then rely on receptor-mediated transcytosismechanisms to deliver the linked protein (e.g., therapeutic protein) tothe cell of interest (e.g., endothelial cells, macrophage or neuronalcells). Physiologically, receptor-mediated transport is relied upon totransport macromolecules (e.g., proteins, into the cell, and generallyinvolves ligand-recognition and binding of a macromolecule (e.g., andIGFI or IGFII moiety) to a specific receptor binding domain (e.g., thecation-independent mannose-6 phosphate receptor (CI-MPR), the IGFIreceptor, the IGFII receptor or the IGFII/CI-MPR) on the targeted cells.Following recognition and binding of the ligand to the binding domain onthe receptor, the receptor-ligand complex undergoes endocytosis by thecell (e.g., endothelial cells or macrophage) and the complex is therebyinternalized. The ligand may then be transported across the abluminalmembrane of the cell (e.g., an endothelial cell, a neuronal cell, aglial cell, a perivascular cell and/or a meningeal cell) and into theappropriate tissue (e.g., tissues of the central nervous system such asbrain or spinal tissue). In certain embodiments described herein, abinding or targeting peptide of a polypeptide composition comprises SEQID NO. 4, amino acid residues 8 through 67 of IGFII.

Also contemplated herein is the inclusion of functional protein labelsor tags into the disclosed compositions to provide additional means ofisolating and/or detecting the translated polypeptide or protein.Suitable labels and tags are well known in the art and, for example,include, but are not limited to luciferase, green fluorescent protein,alkaline phosphatase, horseradish peroxidase, myc-tags, FLAG tags, eTagsand polyhistidine tags. In a preferred embodiment, such labels and tagsare capable of providing a detectable signal to facilitateidentification of such labels or tags, for example upon distribution ofthe amino acid sequence encoding the polypeptide composition into thedesired cells and tissues (e.g., CNS tissue).

The polypeptide compositions described herein which comprise twopolypeptides connected by a linker sequence, can be syntheticallyproduced (e.g., by chemical synthesis) or encoded for and expressed by(e.g., transcribed and translated) a polynucleotide (e.g., nucleic acid)sequence. As used herein, a “polynucleotide” refers to contiguous,covalently linked nucleic acid or nucleic acid molecules, such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides,fragments generated by any appropriate means known in the art (e.g.,ligation or polymerase chain reaction (PCR)), and/or fragments generatedby any of ligation, scission, endonuclease action, and exonucleaseaction. Polynucleotide molecules can be composed of monomers that arenaturally-occurring nucleotides (such as DNA and RNA). In someembodiments, the polypeptide compositions described herein are encodedby the homologous polynucleotide sequences. The polynucleotides areproduced using recombinant DNA technique, known to those with skill inthe art (see, e.g., Sambrook et al., 1992). Generally, in accordancewith the present invention, one or more targeting agents (e.g., aligand/binding protein like IGFII or a biologically active fragmentthereof) are operably linked to a nucleic acid or an amino acid sequenceencoding a therapeutic agent (e.g., the enzyme NaGlu or a biologicallyactive fragment thereof). In some of the polynucleotide moleculesdescribed herein, comprised of nucleotide sequences of at least twogenes joined by a linker sequence, can produce fusion or chimericpolypeptides that may represent a hybrid of the two proteins. Thepolypeptide compositions described herein may also comprise suitableregulatory elements which can be cloned into an expression vector andexpressed in a suitable host. Recombinant methods for designing,expressing and purifying fusion proteins are known in the art (see, e.g.Sambrook, et al., 1992).

Also contemplated herein are expression vectors containing theabove-described polynucleotides and recombinant cells comprising thepolynucleotides or expression vectors. An “expression vector” is apolynucleotide molecule encoding a gene that is expressed in a hostcell. Typically, an expression vector comprises a transcriptionpromoter, a gene, and a transcription terminator. Gene expression isusually placed under the control of a promoter, and such a gene is saidto be “operably linked to” the promoter. Similarly, a regulatory elementand a promoter can be operably linked if the regulatory elementmodulates the activity of the promoter. A “recombinant” or “host” cellused for expression of a vector is a cell that contains a polynucleotidemolecule, such as the polynucleotides described herein. Large amounts ofproteins may be produced in vitro using such expression vectors and/orrecombinant cells and, accordingly, contemplated herein are methods ofproducing the disclosed polypeptide compositions. Such methods involveculturing a recombinant and/or host cell comprising a polynucleotide(e.g., expression vector) under conditions suitable for expression ofthe polypeptide from the polynucleotide. Any cell with protein syntheticcapacity may be used for this purpose (e.g., animal, bacterial, yeast orinsect cells). If a particular protein modification is required, animalcells and, in particular, mammalian cells may be necessary. Cells thatmay be used to express the polypeptide compositions include, but are notlimited to, HT1080, HF1156, Chinese hamster ovary (CHO) cells, CHO-K1cells, HeLa cells, Vero cells, FAO (liver cells), human 3T3 cells, A20cells, ETA cells, HepG2 cells, J744A cells, Jurkat cells, P388D1 cells,RC-4B/c cells, SK-N-SH cells, Sp2/mIL-6 cells, SW480 cells, 3T6 Swisscells and the like. Suitable conditions for protein expression invarious cells/systems are dependent on the cells/system and well-knownin the art (Sambrook et al., 1992).

Also contemplated are pharmaceutical compositions that can beadministered to a subject (e.g., a subject with a disease or disorder)to achieve a desired therapeutic effect (e.g., distribution into thecells and tissues of interest). Pharmaceutical compositions contemplatedherein include, for example, nucleic acid or amino acid sequencesencoding one or more therapeutic agents (e.g., the lysosomal enzymeNaGlu), operably linked to one or more targeting ligands (e.g., afragment of IGFII), or vector or cells comprising the nucleic acid oramino acid sequences. Such amino or nucleic acids may be administeredalone, but are preferably administered in combination with at least oneother agent or excipient (e.g., a pharmaceutically-acceptable carriersuch as buffered saline, dextrose, and purified water).

Suitable pharmaceutically-acceptable carriers preferably stabilize theproteins, enzymes, nucleic acids, amino acids and/or polypeptidessuspended or solubilized therein and facilitate the processing of suchproteins, enzymes, nucleic acids, amino acids and/or polypeptides intopharmaceutical compositions which may be administered to a subject. Thedescribed pharmaceutical compositions can be administered by any numberof routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.

Pharmaceutical formulations suitable for parenteral administration canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as physiologically buffered saline. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension also can containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Further details on techniques for formulation and administration can befound in the latest edition of Remington's Pharmaceutical Science (MaackPublishing Co., Easton, Pa.). After pharmaceutical compositions havebeen prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition (e.g., for the treatmentof Sanfilippo syndrome). Such labeling may include, but not be limitedto instructions to calculate an effective amount of the pharmaceuticalcomposition to be administered to the subject, appropriate dosingschedules, acceptable routes of administration and anticipated adverseeffects.

Also contemplated are methods of delivering a therapeutic polypeptide toa subject in need thereof by administering the polypeptide compositionsand/or expression vectors and/or recombinant cells disclosed herein, toa subject in need thereof. Further contemplated are methods of treatinga lysosomal storage disorder (e.g., Sanfilippo syndrome) byadministering an effective amount of the disclosed polypeptidecompositions to a subject in need thereof. As used herein, the term“subject” is meant to refer to any mammal (e.g., human, mouse, rat, dog,cat, pig, monkey, horse), particularly humans. In certain embodiments,the subject is an adult, an adolescent or an infant. Also contemplatedby the present invention is the administration of the compositionsand/or performance of the methods of treatment in-utero. Thecompositions and methods disclosed herein may be administered using anyof the above-described routes of administration and, in certainembodiments, the polypeptide compositions are administered parenterally.

As used herein, the phrase “effective amount” refers to the amount oftherapeutic agent and/or polypeptide needed to achieve a desiredclinical effect and is largely determined based on the total amount ofthe therapeutic agent contained in a pharmaceutical composition.Generally, an effective amount of a therapeutic agent is sufficient toachieve a meaningful benefit to the subject (e.g., treating, modulating,curing, preventing and/or ameliorating the underlying disease orcondition). For example, an effective amount of a pharmaceuticalcomposition described herein may be an amount sufficient to achieve adesired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disorder or the symptoms thereof.Generally, the amount of a therapeutic agent (e.g., a recombinantlysosomal enzyme) administered to a subject in need thereof will dependupon the characteristics of the subject. Such characteristics includethe condition, general health, age, sex and body weight of the subject.One of ordinary skill in the art will be readily able to determine theappropriate dosages depending on these and other related factors. Inaddition, both objective and subjective assays may optionally beemployed to identify optimal dosage ranges.

EXEMPLIFICATION

To maximize uptake of the lysosomal enzymeN-acetyl-alpha-glucosaminidase (Naglu), a cassette encoding residues8-67 of the mature IGFII was fused in frame to the C-terminus of thefull length human Naglu open reading frame. The design of this constructwas similar glucoronidase-IGFII fusion protein described by LeBowitz etal. (PNAS 101(9):3083-3088, 2004), who observed that cellular uptake ofglucoronidase, through the IGFII cation-independent mannose-6-phosphatereceptor, was greatly improved when fused to IGFII, which binds withhigh affinity to a distinct site on the receptor. To establish thebenefit of such a fusion protein, two expression plasmids were generated(FIG. 3A), one expressing full length wild-type human Naglu and theother expressing the Naglu-IGFII fusion protein with a linker regionconsisting of 3 residues, glycine alanine praline (GAP) (LeBowitz,2004).

HT1080 mammalian stable cell lines were generated for both wild-typeNaglu and Naglu-IGFII. Following seeding of stable cell lines at 1×10⁶cells/ml and culture at 33° C. for 24 hours, protein expression wasmonitored in the conditioned media by western blot and activitydetermined by measuring the cleavage of the fluorogenic substrate4MU-N-acetyl-alpha-D-glucosaminide. It was determined that activity,normalized by cell number, of Naglu-IGFII was 10 fold lower than thatobserved for untagged Naglu (see FIG. 9). By western blot, when sampleload was normalized by cell number, it was apparent that Naglu-IGFIIexpression was significantly less than untagged Naglu (FIG. 10).Furthermore, there was also a significant amount of degradationoccurring during Naglu-IGFII expression as evidenced by the lowermolecular weight band running beneath the upper band in the Naglu-IGFIIlane of the western blot (arrow, FIG. 10). This level of expression andbreakdown was observed in all Naglu-IGFII stable clones developed whichencompassed 3 separate stable transfections. In fact, when analyzingexpression level and activity of various Naglu-IGFII clones, there was avery obvious correlation between the amount of degradation observed bywestern blot and the level of Naglu-IGFII activity in the sample. Thus,clones displaying higher levels of Naglu-IGFII in the conditioned mediatypically had a corresponding higher level of degradation on westernblots. It was suspected that the Naglu-IGFII was relatively inactive dueto the placement of the IGFII protein, and that the level of activityobserved in samples taken from Naglu-IGFII clones was primarily due toclipping of the IGFII protein, resulting in an increased level ofactive, untagged Naglu in the cell-conditioned media samples.

It was hypothesized that increasing the linker length between Naglu andIGFII might improve the expression and activity of the Naglu-IGFIIfusion protein by allowing for a more conformationally stable foldedprotein and/or prevent any potential interference between IGFII and theNaglu active site. To increase the linker length, complementary oligoswere generated consisting of glycine alanine glycine repeats flanked bythe original gap linker, which is encoded, by an AscI restrictionendonuclease site. The oligo was then ligated into the AscI site. Inthis ligation, 3 gag oligos were incorporated into the linker, resultingin a linker that was 57 amino acids long (FIG. 3B and FIG. 8). Stableclones generated from this construct yielded Naglu-GAG₃-IGFII proteinthat was as active (FIG. 9) and expressed to similar levels (FIG. 10) asNaglu. This confirmed that a longer linker allowed for proper foldingand enzymatic activity of the Naglu-IGFII fusion protein. Furthermore,recombinant Naglu-GAG₃-IGFII and Naglu were tested for uptake in humanfibroblast cells (HF1156). Naglu-GAG₃-IGFII was readily taken up by thecells, and that uptake was inhibited by IGFII but not mannose6-phosphate (M6P), in a dose-dependent manner (FIG. 11). As expected,recombinant Naglu, lacking M6P, showed no uptake in the cells (FIG. 11).

Thus, the inventors surprisingly discovered a peptide linker whichresulted in a highly expressed and active NaGlu-IGFII polypeptide. Fromthis work, it is believed that a longer linker (e.g., longer than 3amino acids), resulted in the proper folding/activity of the protein andprevented its degradation. Accordingly, this and similar peptide linkerscan be used generally to link other proteins and create heterologouspolypeptides.

While certain compositions and methods of the present invention havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds of theinvention and are not intended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications, websites and other reference materials referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference.

What is claimed is:
 1. An isolated polypeptide composition comprising: a) a first peptide; b) a second peptide; and c) a linker comprising one or more sequential repeats of the amino acid sequence SEQ ID NO: 1 disposed between said first peptide and said second peptide.
 2. The polypeptide composition of claim 1, wherein said linker comprises three sequential repeats SEQ ID NO:
 1. 3. The polypeptide composition of claim 1, wherein said linker further comprises the amino acid sequence glycine-alanine-proline (GAP) at the C-terminus of SEQ ID NO:
 1. 4. The polypeptide composition of claim 1, wherein said first peptide comprises the amino acid sequence of SEQ ID NO:
 4. 5. The polypeptide composition of claim 1, wherein said second peptide comprises a receptor binding domain.
 6. The polypeptide composition of claim 1, wherein said second peptide comprises the amino acid sequence of SEQ ID NO:
 6. 7. The polypeptide composition of claim 1, wherein said linker comprises the amino acid sequence of SEQ ID NO:2 disposed between said first peptide and said second peptide.
 8. An isolated polypeptide linker comprising the amino acid sequence of SEQ ID NO:
 1. 9. The polypeptide linker of claim 8, wherein the linker comprises the amino acid sequence of SEQ ID NO:
 2. 10. The polypeptide linker of claim 8, wherein said linker comprises three sequential repeats of SEQ ID NO:
 1. 11. The polypeptide linker of claim 10, wherein the C-terminus of said polypeptide linker further comprises the amino acid sequence glycine-alanine-proline (GAP).
 12. The polypeptide composition of claim 1, wherein said composition further comprises an acceptable carrier. 